We study the mechanism by which transcription is regulated by activators, repressors, terminators, and anti-terminators by using the galactose opern in Escherichia coli as an experimental system. The gal operon is transcribed by two tandem promoters, P1 and P2, which are regulated in different ways by several regulatory proteins. We report several discoveries made this year. Studies on Requirements of DNA Looping We previously showed that HU binds to the apex of the gal DNA loop at affinities much higher than its well characterized nonspecific DNA binding. This high affinity binding was totally dependent upon the binding of GalR to the two operators (cooperativity). Now we showed that the cooperativity is the result of a specific GalR-HU interaction. The interaction was demonstrated by co-immunoprecipitation, as well as by isolation and characterization of interaction defective HU mutants. We concluded that GalR "piggybacks" HU to the HU binding site for DNA looping. We studied the GalR/HU mediated DNA looping by atomic force microscopy and confirmed that looping occurs by an "antiparallel" not a "parallel" geometry of the two GalR binding sites in DNA. We identified the GalR dimer-dimer interaction interface during DNA looping by genetic analysis. A set of five amino acid residues, located in a dimer surface within a "crescent" area were identified by isolating and characterizing mutants of GalR that bind to DNA but do not tetramerize. We previously showed that the GalR-GalR interaction to create a DNA loop is facilitated by binding of a DNA bending protein, HU, approximately in the apex of the loop. The role of HU in DNA looping was shown only to stabilize the GalR tetramerization. We isolated and characterized GalR mutants, which form stronger tetramers and which do not need HU to loop and repress, confirming that the role of HU is only to aid and is not essential. Role of -11 Base Pair in Promoter"Melting" We identified the omnipresent adenine at the -11 position of the non-template strand of a promoter as the "master" base in DNA strand separation. The A:T base pair at this position distorts first. Mutational changes at this site prevent strand separation everywhere else in the promoter. We further showed an unsubstituted C2 hydrogen of a purine base is critical and sufficient at the -11 master position to signal base pair deformation. Transcription repression by inhibition of promoter clearance by the activator protein CRP: CRP stimulates transcription from the lac promoter by binding to a site that is -61 bp upstream of the transcription start point. As mentioned above, we isolated a CRP independent lac promoter variant that showed a strong activity. We converted this strong lac promoter, which does not need CRP for transcription, into an extended -10 promoter type by setting a 5'TG3' sequence one base pair upstream of the -10 region. We discovered that CRP binding to the -61 site repressed transcription from the extended -10 promoter. Analysis of transcription products demonstrated that CRP blocks the step of promoter clearance. In the presence of CRP, productive RNA synthesis decreased and abortive RNA synthesis increased. This finding is consistent with our previously proposed model that a regulator acts by differential contacts with RNA polymerase. In this case, CRP contact with RNA polymerase lowers the activation energy to reach the transition state of the "idling" complex to the elongating complex. Effect of DNA supercoiling on transcription initiation Transcription of many genes, like other DNA transactions, is affected by DNA supercoiling. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such affects are exerted were not systematically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters, including the P1 and P2 promoters of gal, present on a plasmid by using a series of its topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays performed on these topoisomers to study full-length and abortive (when applicable) RNA synthesis in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter-to-promoter. Among those promoters, which included P1 and P2, in which DNA superhelicity stimulated transcription, some displayed specific optima of superhelical density while others did not. The results also showed that the amounts of abortive transcripts made from the two gal promoters decreased with increased negative supercoiling, suggesting for the first time an inverse relationship between full-length and abortive RNA synthesis, thus, supporting a role of DNA superhelicity in promoter clearance. The effect of varying amount of supercoiling on the action of gene regulatory proteins, CRP and GalR, suggested mode of action (both activation and repression) of the respective regulators, which are consistent with previous models of their action. Our results underscored the importance of DNA supercoiling in fine tuning of promoter activities which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments.